1. Field of the Invention
The present invention relates to a method of detecting mutation in the base sequence of nucleic acid including DNA (deoxyribonucleic acid) and RNA (ribonucleic acid).
2. Description of the Prior Art
It has been clarified that many cancers and genetic diseases are caused by mutation in the base sequence of DNA. The mutation in the base sequence is generally monobasic substitution. A number of methods have been proposed in the technical field of detecting such mutation in the base sequence. Some of the methods shall now be illustrated.
1) DNA (RNA) Sequencing:
The base sequence of a substance to be analyzed is directly analyzed and decided.
2) DNA Chip:
A number of oligonucleotides are fixed onto a glass surface and selectively hybridized with a substance to be analyzed such as a DNA fragment for thereafter detecting a signal based on the hybridization, generally a fluorescent signal, and comparing the same with a normal one thereby estimating the sequence of the substance.
3) SSCP (Single Strand Conformation Polymorphism) Method:
Double stranded DNA (RNA) employed as a sample is dissociated into single stranded DNA for thereafter detecting difference of the higher-order structure of the single stranded DNA, having a specific higher-order structure depending on the base sequence, by polyacrylic amide gel electrophoresis through difference of mobility depending on the higher-order structure, thereby estimating presence/absence of monobasic substitution.
4) DGGE (Denaturing Gradient Gel Electrophoresis) Method:
A sample of a PCR (polymerase chain reaction) product is electrophoresed in a polyacrylic amide gel formed with a concentration gradient of a denaturant for comparing dissociation from double stranded DNA into single stranded DNA at a migration speed and detecting presence/absence of monobasic substitution in the sample.
5) DHPLC (Denaturing High Performance Liquid Chromatography) Method:
Sample double stranded DNA and standard double stranded DNA having standard base sequence with respect to its inspected site are mixed with each other, thermally denatured to be dissociated into single strands and thereafter cooled to be re-bonded to double strands. When the sample double stranded DNA has standard base sequence, only a homoduplex having hydrogen bonds formed on all corresponding bases is formed. When monobasic substitution is present on the inspected site of the sample double stranded DNA, a homoduplex and a heteroduplex having a mismatch site formed with no hydrogen bond between parts of corresponding bases are formed. The heteroduplex has a smaller number of hydrogen bonds than the homoduplex. Therefore, presence of the heteroduplex is detected with a high-speed liquid chromatograph through the fact that the melting temperature (Tm temperature: temperature at which 50% of the total concentration of double stranded DNA is denatured to single stranded DNA) of the heteroduplex is lower than that of the homoduplex, for detecting presence/absence of monobasic substitution.
However, the aforementioned conventional methods have the following disadvantages:
1) Although the DNA sequencing is most reliable, a high cost is disadvantageously required for a series of operations. Further, a large-scale automation line is necessary for improving the throughput.
2) The DNA chip itself is extremely high-priced and the number of oligonucleotides fixed onto the chip must be varied with the substance, disadvantageously leading to a high cost.
3) and 4) In each of the SSCP method and the DGGE method, electrophoretic conditions must be studied every sample, while the composition of the electrophoretic gel must also be studied every sample in the DGGE method. Furthermore, in each of these methods, it is disadvantageously difficult to improve the throughput due to the employment of gel electrophoresis.
5) The DHPLC method disadvantageously requires a high-priced liquid chromatograph.
An object of the present invention is to provide a method of detecting mutation in the base sequence of nucleic acid capable of performing high-throughput analysis with no requirement for a high-priced apparatus.
The present invention comprises the following steps (A) to (C) for detecting mutation in the base sequence of nucleic acid:
(A) heating a solution containing double stranded nucleic acid employed as a sample for dissociating the double stranded nucleic acid into single stranded nucleic acid, and cooling and rebonding the same into double stranded nucleic acid,
(B) increasing the temperature of the solution after completion of the step (A) until the double stranded nucleic acid is dissociated into single stranded nucleic acid for measuring ultraviolet absorption of the solution and acquiring a thermal melting profile, and
(C) determining presence/absence of mutation in the base sequence of the double stranded nucleic acid on the basis of the thermal melting profile.
Throughout the specification, the term xe2x80x9cdouble stranded nucleic acidxe2x80x9d includes a DNA/DNA double strand (a double strand of single stranded DNA; referred to as double stranded DNA), an RNA/DNA double strand (a double strand consisting of single stranded RNA and single stranded DNA), an RNA/RNA double strand (a double strand of single stranded RNA) and fragments thereof.
The foregoing and other objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of the present invention.